Revista da Sociedade Brasileira de Medicina Tropical 48(4):410-416, Jul-Aug, 2015 Major Article http://dx.doi.org/10.1590/0037-8682-0185-2015 Detection of immunogenic proteins from Anopheles sundaicus salivary glands Yunita Armiyanti[1], Mohammad Mirza Nuryady[2], Renam Putra Arifi anto[2], Elisa Nurmariana[2], Kartika Senjarini[2], Loeki Enggar Fitri[3] and Teguh Wahju Sardjono[3] [1]. Department of Parasitology, Faculty of Medicine, Jember University, Jember, Indonesia . [2]. Department of Biology, Faculty of Mathematic and Natural Sciences, Jember University, Jember, Indonesia. [3]. Department of Parasitology, Faculty of Medicine, University of Brawijaya, Malang, Indonesia. ABSTRACT Introduction: The saliva of mosquitoes has an important role in the transmission of several diseases, including malaria, and contains substances with vasomodulating and immunomodulating effects to counteract the host physiological mechanisms and enhance pathogen transmission. As immunomodulatory components, salivary gland proteins can induce the generation of specifi c IgG antibodies in the host, which can be used as specifi c biomarkers of exposure to Anopheles sundaicus. The objective of this study was to identify immunogenic proteins from the salivary glands of Anopheles sundaicus by reaction with sera from individuals living in malaria-endemic areas who are thus exposed to Anopheles mosquitoes. Methods: IgG antibodies targeting salivary gland proteins in serum samples from individuals living in malaria-endemic areas were measured by enzyme-linked immunosorbent assay (ELISA). Sera from healthy individuals living in non-endemic areas were used as negative controls. Determination of the presence of salivary gland immunogenic proteins was carried out by western blotting. Results: Sixteen bands appeared in sodium dodecyl sulfate polyacrylamide gel electrophoresis, with molecule weights ranging from 22 to 144kDa. Among the exposed individuals, IgG responses to salivary gland proteins were variable. Protein bands with molecular weights of 46, 41, 33, and 31kDa were the most immunogenic. These immunogenic proteins were consistently recognized by pooled serum and individual samples from people living in malaria-endemic areas but not by negative controls. Conclusions: These results support the potential use of immunogenic proteins from the salivary glands of Anopheles as candidate markers of bite exposure or in malaria vaccines. Keywords: Anopheles sundaicus. Salivary glands. Immunogenic proteins. Antibody. in coastal area of Java and the Sumatra islands. The distribution INTRODUCTION of Anopheles sundaicus also includes countries in Southeast Asia and India(2) (3). Malaria is a mosquito-borne disease that has major health The important role of Anopheles mosquitoes as malaria implications worldwide, with an estimated 207 million vectors is supported by the presence of salivary glands in individuals affected by malaria and 627,000 malaria-related female Anopheles mosquitoes. Anopheles mosquito salivary deaths reported. In 2013, 104 countries and territories, including glands secrete substances that can enhance the transmission of Indonesia, had malaria, which was considered endemic; an Plasmodium(4). These substances inhibit hemostatic processes estimated 3.4 billion people are at risk of contracting malaria(1). and modulate the host immune response (immunosuppressive) Malaria is caused by fi ve species of parasites from the at site of biting, allowing the mosquito to feed on blood genus Plasmodium and naturally spread from one person to successfully(5) (6). These changes at the site of the bite, owing to another by female Anopheles mosquitoes. There are about 400 the effects of salivary substances, would benefi t the pathogen, different species of Anopheles mosquitoes, but only 30 of these permitting infection without any resistance and thus enhancing are vectors of major importance(1). Anopheles sundaicus is one infectivity in the vertebrate host(7). of the most important malaria vectors in Indonesia, particularly Injection of saliva into the host’s skin also induces the production of antibodies against salivary proteins(8). The immunogenicity of salivary proteins has been demonstrated Corresponding author: Yunita Armiyanti. Department of Parasitology/ in previous studies, which showed that salivary proteins can Faculty of Medicine/Jember University. Jember 68121, Indonesia. induce the host immune response by producing immunoglobulin Phone: 62 857 3248-2300 email: [email protected] G (IgG) and immunoglobulin E (IgE) via hypersensitivity (9) Received 19 June 2015 reactions . The presence of a specific IgG antibody Accepted 15 July 2015 response against whole saliva extracts (WSEs) of Anopheles 410 Armiyanti Y et al. - Anopheles immunogenic protein mosquitoes can be measured by enzyme-linked immunosorbent glands were dissected using fi ne entomological needles under a assays (ELISAs) in children and adults exposed to mosquitoes stereoscopic microscope at 4× magnifi cation and collected into bites(10) (11). Additional studies have shown that the IgG response a microcentrifuge tube containing a small amount of phosphate- can recognize specifi c salivary proteins(12) (13). Therefore, salivary buffered saline [(PBS); pH 7.2] and phenylmethylsulfonyl proteins (e.g., gSG6) that are able to generate specifi c IgG fl uoride (PMSF) as protein inhibitors. The salivary glands were antibodies have been developed as serological indicators or stored at -80°C until use. biomarkers of exposure to malaria vectors(14). Salivary gland extraction and protein quantifi cation Immunoglobulin G antibodies against Anopheles salivary proteins in the host may be associated with protection against One hundred salivary glands pairs in PBS and PMSF were malaria. In malaria-endemic regions, populations exposed thawed on ice and mixed in 1:1 lysis buffer containing 1.5mM to uninfected Anopheles bites repeatedly over the years may MgCl2, 10mM Tris HCl, 10mM NaCl, 1% Nonidet P-40, and develop an anti-saliva immune response by producing specifi c 2mM ethylenediaminetetraacetic acid (EDTA) NaOH(20). The antibodies in the presence of interleukin (IL)-10. These specifi c mixture was homogenized using a micropestle and sonicated antibodies will neutralize some salivary proteins of vectors, using a water sonicator for 30 min. After centrifugation thus causing micro-environmental changes at the site of the (12,600rpm for 15 min at 4°C), the resulting supernatant was mosquito bite and ultimately affecting malaria transmission. collected and concentrated using a spin concentrator (cut-off of Therefore, in asymptomatic patients with malaria, the level of 10kDa; Corning) and centrifugation (10,000rpm for 30s at 4°C). IgG antibodies targeting anti-salivary gland sonicates (SGSs) The protein concentrations of SGEs were determined using a from Anopheles darlingi are higher than those in symptomatic nanophotometer (Nanophotometer Implen P 360, Germany). patients and healthy individuals(15). Exposure to Anopheles Salivary gland extracts were then diluted in 0.1M bicarbonate mosquito bites and Plasmodium infections, which persist for a buffer (pH 9.6) to obtain a protein concentration of 1µg/µL for long time, affect the development of the immune response to enzyme-linked immunosorbent assay (ELISA)(11). parasites and salivary vectors, thereby infl uencing the number Human serum samples of parasites and the host response(16). Thus, salivary components could be effective vaccine candidates for reducing the morbidity Twenty serum samples were collected from healthy of vector-borne diseases through combination with other malaria adult residents living in the location at which we collected vaccine candidates to protect against severe malaria(7). Further An. sundaicus, i.e., Bangsring village, Wongsorejo District, characterization of salivary proteins and the immune response Banyuwangi Regency in East Java province, to detect antibodies is needed to identify salivary proteins involved in protection against An. sundaicus salivary gland proteins. Seven serum against malaria; the fi rst step in this process is determination samples from healthy adult residents living in non-malaria of the immunogenicity of the salivary proteins. Many studies regions were used as negative control. The human subjects have been conducted to identify and characterize immunogenic protocol for this study was approved by the Ethical Committee salivary proteins from malaria vectors in Africa; however, of Medical Research, Medical Faculty, Brawijaya University. malaria vectors in Asia, particularly An. sundaicus, have not ELISA been studied(8) (17) (18). Therefore, in this study, we measured the levels of IgG To optimize the working conditions for ELISAs, checkerboard antibodies targeting salivary proteins in serum samples from titration was carried out using An. sundaicus SGEs at 1, 2, and individuals living in malaria-endemic areas using salivary 4µg/mL and serially diluted serum samples (1:25, 1:50, and glands extracts (SGEs) from An. sundaicus. Based on the 1:100) from healthy individuals living in the malaria-endemic IgG antibody response to salivary proteins, we identifi ed the region. ELISA was performed as described by Fontaine immunogenic proteins contained within An. sundaicus SGEs. et al.(11). Based on the results of checkerboard titration, microtiter immunoplates (SPL Life Sciences, Korea) were coated with 4µg/ml (50µL/well) of An. sundaicus SGEs diluted in 0.1M METHODS bicarbonate buffer (pH 9.6) overnight at 4°C. Three washes